Method for differential diagnostics of viral gastro-intestinal infections in cattle due to immunoenzymatic assay

FIELD: veterinary virology.

SUBSTANCE: the present innovation deals with interaction of antibodies with an antigen, with antibodies labeled with horseradish peroxidase, addition of substrate mixture and registration of reaction results. Moreover, one should apply plotting boards with presorbed antibodies and general immunoenzymatic conjugate. The innovation enables to shorten terms for diagnosis and obtain more significant results of diagnostics.

EFFECT: higher efficiency.

 

The present invention relates to the field of veterinary Virology, in particular to the development of a method for differential diagnosis of viral gastrointestinal infections bovine enzyme-linked immunosorbent assay.

A leading role in the etiology of diarrhea in young farm animals play Rota-coronavirus, virus diarrhea - diseases of the mucous membranes, to a lesser extent other infectious agents. These diseases are widely spread in the Russian Federation and abroad, and put livestock farms tangible economic damage resulting from the loss from death, especially in the early years, funds spent on treatment of patients, and loss of production from the animals recover. Every year half of the losses of juveniles accounted for gastrointestinal disease at an early age.

With great efficiency to solve the problem of differential diagnosis allows a highly sensitive enzyme immunoassay.

In connection with the above, problem diagnosis and control of these infections is important.

A known method for the diagnosis of viral gastrointestinal infections bovine enzyme-linked immunosorbent assay using a diagnostic kit [1].

Also there is a method of detection of antig is on virus diarrhea what specific immunoglobulins included in a diagnostic kit, making wells at 0.1 ml and adsorb on their surface. The solution containing the investigated antigen, contribute to the wells at 0.1 ml and incubated with sensitized on the surface of the carrier antibodies for 1.5-2 h at 37°C. Unbound antigen is removed by washing. Then add enzyme conjugate in a volume of 0.1 ml In positive cases, the conjugate binds fixed on the sensitized surface antigen, forming a complex of the antibody-antigen-conjugate. The excess conjugate is removed by washing and contribute to the wells of the substrate indicator solution, which changes its color according to the principle of redox reaction, is proportional to the number of conjugated with antibodies to the enzyme. The amount of enzyme is proportional to the amount of antigen fixed on the sensitized surface of the hole of the die [2].

The objective of our research was to develop a more reliable method of differential diagnosis Rota-coronavirus enteritis, viral diarrhea, bovine enzyme-linked immunosorbent assay with a significant reduction in time to diagnosis.

The essence of the proposed method consists in the following.

To the wells of one plate with previtellogenesis antirotavirus, anticolonialism, antidiarrheal immunoglobulins automatic pipette make a test sample by 0,10-0,12 ml in the wells with different antibodies, incubated for 1.5-2 hours at a temperature of 37-38°C, washed tablet, contribute 0,10-0,12 ml total enzyme conjugate, consisting of labeled enzyme antibodies to these pathogens, and incubated under the same conditions, the tablet is washed and after addition of the substrate mixture is conducted concurrently taking into account the results on Rota-coronavirus enteritis, viral diarrhea, bovine enzyme-linked immunosorbent assay.

Example 1.

In the first stage reaction in all wells automatic pipette contribute to 0.1 ml of a phosphate buffer solution. In the first wells of rows with rotavirus, coronavirus, diarrhoeal immunoglobulins contribute to 0.1 ml of the test sample, pipeinput, and 0.1 ml is transferred into the next pit, etc. To control reactions in parallel contribute to 0.1 ml of specific homologous antigens and also titrated them as a sample. In the same way make a negative antigen. The tablet is incubated in the incubator for 1.5-2 h at 36-37°C, then washed from unbound antigen. In the washed wells contribute to 0.1 ml of the total enzyme conjugate, consisting of antirotavirus, anticorrosiveness, antidiarrheal immunoglobulins, uchenykh horseradish peroxidase. Tablet incubated in a thermostat at 36-37°1.5-2 h and again washed three times with phosphate-buffer solution. Then in the wells contribute to 0.1 ml of substrate mixture consisting of a solution of 5-aminosalicylic acid and hydrogen peroxide. The reaction account for 30-40 min by a colour change of the formed reaction product from brown to light brown

- intense brown staining, brown staining - positive result;

- light brown staining - equivocal result;

- staining is absent or is at the level of the negative control is a negative result.

The proposed method, in contrast to the known, allows for diagnostics Rota-coronavirus enteritis, viral diarrhea in cattle by ELISA significantly reduce the time for setting-up the reaction and to obtain more reliable results when taking into account, as the research on these infections are carried out simultaneously on the same tablet with pre-adsorbed antirotavirus, anticolonialism, antidiarrheal immunoglobulins using a General-enzyme conjugate under the same conditions.

The proposed method for the differential diagnosis of Rota-coronavirus enteritis, viral diarrhea cattle by the method of immunore the elemental analysis is tested by a Commission in October 2001 and approved in some veterinary laboratories with a positive result.

On the basis of the obtained test results, the Commission concluded that the set is active and specific and can detect these specific antigens in the material studied in much shorter time.

The proposed method will be applied for the inspection of cattle affected with gastrointestinal infections farms for the purpose of diagnosis Rota-coronavirus enteritis, viral diarrhea cattle. High sensitivity, specificity, ease of setting reaction and the possibility of automation of the processes of research allows us to quickly carry out mass screening animals to determine the spread of these infections.

Sources of information

1. Proceedings of VIEW, VIP, M. 1984, p.63-66.

2. Reports of the Russian Academy of agricultural Sciences, M. 1998, issue 3, s.

The method of differential diagnosis of viral gastrointestinal infections bovine enzyme-linked immunosorbent assay, mainly Rota-coronavirus enteritis, viral diarrhea in cattle, including the interaction of the antibody with the antigen with antibodies labeled with horseradish peroxidase, the addition of the substrate mixture and records the results on the intensity of the color formed complex, characterized in that the reactions which use tablets with pre-adsorbed on them antirotavirus, anticolonialism, antidiarrheal antibodies and after application of the tested samples 0,10-0,12 ml tablets incubated, washed, and further contribute 0,10-0,12 ml total enzyme conjugate, consisting of enzyme labeled antibodies to these pathogens and then hold the light of the results.



 

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